This invention relates to cell culture inserts for insertion into multi-well plates for culturing cells. More particularly, this invention relates to such inserts which have placed on the bottom surface thereof a microporous membrane as the substrate for cells being cultured. Even more particularly, this invention relates to such a cell culture insert device for supporting that insert in an inverted position initially so that cells may be developed on the bottom surface of the membrane of the support for subsequent interaction of developing cells on both the bottom and top surfaces of the porous membrane.
Initially, when cell inserts were developed for multi-well plates, they were comprised of a plastic material with an amorphous membrane material on the bottom surface thereof, for propagating cells. The inserts were placed in a conventional multi-well plate in the individual compartments therefore. However, in recent years, with the use of a suspended microporous membrane, two cell types for example, can be cultured, one on each side of the membrane in the same well. The microporous membrane allows free passage of macromolecules, proteins and ions. As a result, the interaction of the two cell types can be studied without actual physical contact between the two cell populations in the suspended state of the insert. The growth environment mimics the in vivo state of cells being developed in vitro and may replace in vivo testing which has taken place in the past.
As purely representative of materials which may be utilized for the microporous membranes in the device of the invention here, include, for example, polycarbonate and polyethylene terephthalate. The porosity of the membrane is developed to allow for selective permeation, as discussed above. The membrane material and/or the degree of porosity is developed to allow for direct viewing with phase contrast microscopy, as one feature of the use of such porous membranes. The membranes preferably are transparent or translucent.
As a further feature of this invention, it should be understood that the membrane porosity will be determined by specific applications. A representative porosity may be within the range of, for example, between about 0.2-10 microns.
As discussed above, and as a further background of the invention here, filter well culture on porous membrane substrates is growing in popularity because it provides a defined and reproducible culture environment that resembles the in vivo environment much more closely than ordinary cell culture conditions such as modified surfaces on petri dishes, for example. Some of the advantages include the ability to generate much higher cell densities, the possibility of establishing differentiated cell polarity, direct cell access to medium from below as well as from above, better gas exchange and the possibility of studying heterologous cell interactions that are mediated by diffusable substances.
This new and popular technology is used to enhance cellular differentiation, to study polarized ions and molecular transport across confluent epithelial monolayers, to more easily establish primary cell cultures, to study indirect i.e., diffusable substance-mediated cell interactions and to study the metastatic potential of malignant tumor cells.
Representative prior art includes U.S. Pat. No. 4,308,351 and is representative of current procedures now being used for cell culture inserts. U.S. Pat. No. 4,748,124 teaches a complex reusable culture chamber. Neither patent contemplates culturing on both sides of the same membrane.
In order to do this in the conventional way now, investigators are currently required to aseptically invert a conventional multi-well plate insert and try to establish a cell culture on the flat bottom surface of the membrane in a large drop of liquid medium whose depth is maintained only by surface tension on that flat surface. If these cells attach successfully, then the insert is inverted back to its normal orientation and placed into the multi-well plate containing a medium, wherein the second monolayer is then established inside the filter insert in the conventional manner.
It is to this problem that the present invention is directed so as to remove the improvised technique which often fails because of the compromised sterility or because the initial medium-and-cell droplet penetrates into the membrane too rapidly and/or evaporates prematurely.
Other objects and advantages of this invention will be apparent from the following description, the accompanying drawings and the appended claims.